MPMI Vol. 13, No. 11, 2000, pp. 1177–1183. Publication no. M-2000-0828-01R. © 2000 The American Phytopathological Society
The
sss Colonization Gene
of the Tomato-Fusarium oxysporum f. sp.
radicis-lycopersici Biocontrol Strain Pseudomonas
fluorescens WCS365 Can Improve Root Colonization
of Other Wild-type Pseudomonas spp. Bacteria
Linda C. Dekkers, Ine H. M. Mulders, Claartje C. Phoelich, Thomas F. C. Chin-A-Woeng, André H. M. Wijfjes, and Ben J. J. Lugtenberg
Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, Leiden 2333AL, The Netherlands
Accepted 16 June 2000.
We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp.
radicis-lycopersici can be controlled by inoculation of seeds
with cells of the efficient root colonizer Pseudomonas
fluo-rescens WCS365, indicating that strain WCS365 is a
bio-control strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes en-coding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as micro-colonies of either one or both cell types. Micromicro-colonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in com-parison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consis-tently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engi-neering is a realistic goal.
Commercial application of fluorescent Pseudomonas spp. as biocontrol agents against a large group of plant pathogens
can be hampered by their inconsistent performance in the field (Schroth and Hancock 1982). These inconsistencies are often accompanied by inefficient colonization of the plant root by the biocontrol strain (Schippers et al. 1987), suggesting that colonization is often the limiting factor for biocontrol.
To study colonization, we selected P. fluorescens strain WCS365 (Geels and Schippers 1983a, 1983b) as the most ef-ficient root colonizer among a series of biocontrol strains (Brand et al. 1991). This strain is not antagonistic against a large number of fungal pathogens on antagonistic test plates under laboratory conditions (T. F. C. Chin-A-Woeng, unpub-lished data). However, strain WCS365 induces induced sys-temic resistance (ISR) in Arabidopsis thaliana ecotype Co-lumbia, which thereby is protected against the pathogen P. syringae pv. tomato strain DC3000 (Gerrits and Weisbeek 1996). Therefore, we tested whether P. fluorescens WCS365 is also able to control tomato foot and root rot, the plant dis-ease studied in our previous work (Chin-A-Woeng et al. 1998; Lugtenberg et al. 2000).
In order to unravel the molecular basis of root coloniza-tion, P. fluorescens WCS365 has been used for the genera-tion of colonizagenera-tion mutants (Dekkers et al. 1998a, 1998b, 1998c; Lugtenberg and Dekkers 1999; Simons et al. 1997). One of the colonization mutants, strain PCL1233, was com-plemented for colonization by a wild-type DNA fragment containing a multicistronic transcription unit that comprises at least six open reading frames (ORFs) (Dekkers et al. 1998b). The fifth ORF of this DNA fragment, which is ho-mologous to the site-specific recombinases Sss (Hofte et al. 1994), XerC (Colloms et al. 1990), FimB, and FimE (Dorman and Higgins 1987; Klemm 1986), was shown to be crucial for colonization (Dekkers et al. 1998b). This gene is thought to play a role in DNA rearrangements that regulate the transcription of a gene or a set of genes involved in the biosynthesis of cell surface components (Dekkers et al. 1998b). In a more general perspective, it is thought that lack of DNA rearrangements in mutant PCL1233 results in cells that are locked in a state unfavorable for competitive coloni-zation (Dekkers et al. 1998b).
In this article, we show that WCS365 is able to control to-mato foot and root rot and describe how its colonization be-havior on the tomato root is influenced by the presence of the antibiotic-producing biocontrol strain P. chlororaphis PCL1391. Moreover, we describe the effect of inactivated col genes, in-cluding sss, on biocontrol. Finally, we describe how the intro-duction of the sss operon into other Pseudomonas spp. affects the colonization abilities of these wild-type strains.
RESULTS
Biocontrol by P. fluorescens WCS365 and by a mixture with P. chlororaphis strain PCL1391.
Biocontrol experiments performed in an Fusarium ox-ysporum f. sp. radicis-lycopersici-tomato system showed that P. fluorescens WCS365 (Table 1) in all three experiments suppressed the disease in a statistically significant way (Table 2). P. fluorescens WCS365 showed similar biocontrol activity to PCL1391, a P. chlororaphis strain recently described to
control tomato foot and root rot (Chin-A-Woeng et al. 1998). Considering the observation that both strains occupy similar sites on the tomato root and that both are efficient colonizers (Lugtenberg and Dekkers 1999), we studied how the strains behave on the root after they had been applied on the seedling as a mixture. In order to distinguish the strains, they were ge-netically marked with variants of green fluorescent protein.
Germinated tomato seedlings were inoculated with a 1:1 mixture of red fluorescent (DsRed) or enhanced cyan fluores-cent protein (ECFP)-marked WCS365, and enhanced green (EGFP) or yellow (EYFP) fluorescent protein-marked P. chlororaphis PCL1391 (Bloemberg et al. in press). Using con-focal laser microscopy on 1-week-old roots of sterile tomato plantlets, we observed that bacterial cells are mainly present as elongated stretches on indented areas, such as junctions between epidermal cells and the deeper parts of the epidermis on the root surface and on root hairs (Fig. 1). The highest numbers of bacteria and microcolonies were found at the root base (Fig. 1), whereas numbers of both bacterial cells and mi-Table 1. Bacterial strains and plasmids used in this study
Strains and plasmids Characteristics and references Pseudomonas fluorescens
WCS365 Isolated from potato (Geels and Schippers 1983a, 1983b). Efficient colonizer of potato roots (Brand et al. 1991; Glandorf 1992) and tomato roots ( Simons et al. 1996). Causes induced systemic resistance in Arabidopsis thaliana ecotype Columbia against Pseudomonas syringae pv. tomato (Gerrits and Weisbeek 1996). Bio-control strain in a Fusarium oxysporum f. sp. radicis-lycopersici-tomato system (this article).
WCS307 Isolated from potato (Geels and Schippers 1983b). Poor colonizer of potato roots (Brand et al. 1991; Glandorf 1992) and tomato roots ( Simons et al. 1996).
F113 Biocontrol strain in a Pythium ultimum-sugar beet system (Shanahan et al. 1992). Good colonizer of tomato roots (Simons et al. 1996).
PCL1209 Mutant of P. fluorescens strain WCS365 impaired in the synthesis of the O-antigen of lipopolysaccharide and in competitive tomato root tip colonization ( Dekkers et al. 1998a).
PCL1210 Mutant of P. fluorescens strain WCS365 impaired in competitive tomato root tip colonization, in which a mutation in a two-component system is responsible for the mutant phenotype ( Dekkers et al. 1998a). PCL1232 P. fluorescens WCS365 derivative harboring plasmid pMP5215 ( Dekkers et al. 1998b) used for colonization
and biocontrol experiments (this article).
PCL1233 Mutant of P. fluorescens strain WCS365 impaired in competitive tomato root tip colonization in which a mutation in a site-specific recombinase is responsible for the mutant phenotype ( Dekkers et al. 1998b). PCL1269 Mutant of P. fluorescens strain WCS365 impaired in motility and in competitive tomato root tip colonization
(this article).
PCL1391 Wild-type P. chlororaphis, producing phenazine-1-carboxamide and biocontrol strain of tomato foot and root rot caused by F. oxysporum f. sp. radicis-lycopersici (Chin-A-Woeng et al. 1998).
PCL1500 lacZ derivative of P. fluorescens WCS365, which is as efficient as the parental strain in colonizing the tomato root tip (van der Bij et al. 1996).
PCL1502 PCL1500, a Tn5lacZ (Lam et al. 1990) derivative of P. fluorescens WCS365 harboring the empty vector pWTT2081, which is stable in the rhizosphere (van der Bij et al. 1996).
PCL1510 lacZ derivative of P. fluorescens F113 (this article).
PCL1511 lacZ derivative of P. fluorescens F113 harboring the empty vector pWTT2081 (this article).
PCL1512 Derivative of biocontrol strain P. fluorescens F113, which harbors the sss-containing plasmid pMP5215 (this article).
PCL1516 lacZ derivative of P. fluorescens WCS307 harboring the empty vector pWTT2081 (this article). PCL1517 P. fluorescens WCS307 harboring the sss-containing plasmid pMP5215 (this article).
Escherichia coli
DH5α EndA1 gyrSA96 hrdR17(rK- mK-) supE44 recA1. Used for transformation and propagation of plasmids (Boyer and Roulland-Dussoix 1969).
Fungi
Fusarium oxysporum f. sp. radicis-lycopersici strain ZUM2407
Cause of tomato foot and root rot, IPO-DLO, Wageningen, The Netherlands. Plasmids
pRK2013 Helper plasmid for triparental mating ( Ditta et al. 1980).
pWTT2081 Plasmid stably maintained in the rhizosphere (van der Bij et al. 1996). pMP4641 Stable plasmid pME6010 ( Heeb et al. 2000) containing the ecfp gene. pMP4655 Stable plasmid pME6010 ( Heeb et al. 2000) containing the egfp gene. pMP4658 Stable plasmid pME6010 ( Heeb et al. 2000) containing the eyfp gene. pMP4662 Stable plasmid pME6010 ( Heeb et al. 2000) containing the DsRed gene.
crocolonies gradually decreased in the direction of the root tip, on which low numbers of cells were found (Fig. 1). On the upper part of the root system, larger microcolonies were ob-served. The microcolonies were of both mixed and one cell type (Fig. 1A and B). On the middle part of the root (3 to 7 cm from the top), small microcolonies and single cells were ob-served. Most microcolonies were of one cell type, although a mixed microcolony occasionally was present (Fig. 1D and F). On the root tip, single cells or small groups of cells (two to four) often were observed. These small microcolonies are pre-dominantly of one cell type (data not shown). Starting seed inoculation with equal numbers of cells of WCS365 and PCL1391, microcolonies consisting of WCS365 cells were found approximately five times more abundantly than micro-colonies consisting of PCL1391 cells, especially on the mid-dle part of the root (Fig. 1D). In contrast, root hairs were colonized almost exclusively with PCL1391 cells (Fig. 1E). Equal but very low numbers of WCS365 and PCL1391 cells were found on the root tip (data not shown).
Quorum sensing plays a crucial role in the production of phenazine-1-carboxamide (PCN) by PCL1391 (T. F. C. Chin-A-Woeng, D. van den Broek, G. de Voer, K. M. G. M. van der Drift, J. E. Thomas-Oates, B. J. J. Lugtenberg, and G. V. Bloemberg, unpublished data), and PCN is crucial for bio-control by this strain (Chin-A-Woeng et al. 1998); therefore, we wondered what the effect would be on biocontrol of the observed dilution of PCL1391 cells with WCS365 cells, which do not produce detectable acylhomoserine lactones (T. F. C. Chin-A-Woeng, unpublished data). The presence of WCS365 cells also dilutes the quorum sensing signals, espe-cially on the upper parts of the root. The results of four ex-periments (Table 3) show that inoculation with either PCL1391 or WCS365 was significant in only two out of four experi-ments. In contrast, in all four experiments of mixed inocula-tion, significant biocontrol was observed.
Effect of colonization on biocontrol by WCS365.
Recently, it was shown in our laboratory that root coloniza-tion is a requirement for biocontrol by P. chlororaphis strain PCL1391 (Chin-A-Woeng et al. in press), which exerts its biocontrol action through the production of phenazine-1-carboxamide (Chin-A-Woeng et al. 1998). In order to see whether the same applies for biocontrol by P. fluorescens strain WCS365, which acts through ISR, biocontrol activity of colonization mutants of the latter strain was compared with that of the parental strain. The tested colonization mutants were mutant PCL1210, mutated in a two-component system (Dekkers et al. 1998a); mutant PCL1233, which lacks a site-specific recombinase (Dekkers et al. 1998b); mutant PCL1209, which lacks the O-antigen of lipopolysaccharide [LPS] (Dekkers et al. 1998c); and the nonmotile mutant PCL1269. In all seven experiments, the wild-type strain WCS365 showed significant biocontrol (Table 4). Of the tested colonization mu-tants, strain PCL1209 consistently caused biocontrol. The other three mutants caused biocontrol in some, but not all, ex-periments (Table 4).
Effect of extra copies of the sss-containing colonization operon on competitive root tip colonization
of Pseudomonas wild-type strains.
In a second attempt to test a role of colonization genes in the biocontrol ability of strain WCS365, we increased the copy
num-ber of the sss-containing colonization operon by incorporation of this DNA fragment into the rhizosphere-stable plasmid pWTT2081 (four to eight copies). The increase in copy number had no significant effect on the competitive colonization ability of P. fluorescens WCS365 (Table 5). however, the increase in copy number resulted in a statistically significant 8- to 16-fold increase in the colonization ability of P. fluorescens F113 (Table 5) and even in a 13- to 40-fold increase for P. fluorescens WCS307 (Table 5). Strains WCS307 and F113 contain DNA fragments that hybridize with the sss-containing fragment of WCS365. We ex-plain the result by assuming that the majority of the cells of WCS365 are in the rhizosphere-competent state, in contrast to the situation in the other two strains. Multiple copies would result in an equilibrium situation and, therefore, cause poorer colonization of WCS365 and better colonization of the other two strains. DISCUSSION
Biocontrol experiments performed in an F. oxysporum f. sp. radicis-lycopersici-tomato system showed that disease suppres-sion by P. fluorescens WCS365 was statistically significant to a similar extent as P. chlororaphis strain PCL1391 (Table 2). These results show that WCS365 not only is an excellent colo-nizer but also an excellent biocontrol strain. P. fluorescens WCS365 is not antagonistic against phytopathogenic fungi un-der laboratory conditions; therefore, the mechanism unun-derlying biocontrol is likely to be of another nature. Indeed, it has been reported that P. fluorescens strain WCS365 can induce ISR in A. thaliana ecotype Columbia, which thereby is protected against the pathogen P. syringae pv. tomato (Gerrits and Weisbeek 1996). Whether ISR is also responsible for biocontrol in the F. oxysporum f. sp. radicis-lycopersici-tomato system is not known with certainty.
Table 3. Comparison of mixed and single inoculation of tomato with Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391 on tomato foot and root rotz
Diseased plants (%)
Treatment Exp. 1 Exp. 2 Exp. 3 Exp. 4
Untreated control 69 a 60 a 61 a 50 a
PCL1391 52 ab 41 b 47 bc 47 a
WCS365 58 ab 35 b 35 cd 54 a
PCL1391/WCS365 45 b 45 b 25 d 33 b
zData are the result of eight replicates of 12 plants each. Data were analyzed for significance using analysis of variance followed by Fischer’s least significant difference test (α = 0.05), using SPSS soft-ware (SPSS Inc., Chicago). Values followed by different letters indica-tions indicate a statistically significant difference.
Table 2. Biocontrol activity of Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391 in a Fusarium oxysporum f. sp. radicis-lycopersici-tomato systemz
Diseased plants (%)
Treatment Exp. 1 Exp. 2 Exp. 3
Untreated control 45 a 43 a 72 a
WCS365 19 b 25 b 39 b
PCL1391 21 b 27 b 38 b
To test how two Pseudomonas biocontrol strains, which use different mechanisms of action, behave on the root upon in-oculation of the seedlings with a 1:1 mixture of cells of these strains, the two cell types were labeled with different fluores-cent markers. The results (Fig. 1) show that, although the strains use similar overall colonization strategies (Chin-A-Woeng et al. 1997), their precise behavior differs. Cells of strain WCS365 were more abundant at the middle part of the root (Fig. 1D), whereas cells of PCL1391 were more abundant on root hairs (Fig. 1E). Another striking observation was the frequent occurrence of microcolonies of a single cell type, es-pecially at the lower part of the root system. This suggests to us that initial colonization of new root parts occurs predomi-nantly by single cells.
Mixed inoculation with the two cell types tended to give slightly better protection against the disease than single in-oculation with the same number of cells (Table 3). Production of PCN by strain PCL1391 is subject to quorum sensing (T. F. C. Chin-A-Woeng, D. van den Broek, G. de Voer, K. M. G. M. van der Drift, J. E. Thomas-Oates, B. J. J. Lugtenberg, and G. V. Bloemberg, unpublished data). Although it is not known how many cells are required to allow PCN production, the fact that most microcolonies on the lower root parts consist of one cell type (Fig. 1) may contribute to the slight synergistic effect of the two strains in plant protection (Table 3).
Although an important role for colonization in biocontrol was suggested previously (Bull et al. 1991; Schippers et al. 1987), this notion is not general (Roberts et al. 1994). Now that well-characterized colonization-impaired mutants are avail-able (Dekkers et al. 1998a, 1998b, 1998c; Lugtenberg and Dekkers 1999; Lugtenberg et al. 1996; Simons et al. 1996, 1997; Weller 1988) it is possible to test the influence of colo-nization on biocontrol experimentally.
Chin-A-Woeng et al. (Chin-A-Woeng et al. in press) have constructed colonization mutants in P. chlororaphis strain PCL1391 whose PCN production is required for biocontrol of tomato foot and root rot. Individual derivatives of this strain, impaired in sss/xerC, motility, and the synthesis of the amino acid phenylalanine, appeared not to be able to control disease in
an F. oxysporum f. sp. radicis-lycopersici-tomato biocontrol system. Controls show that the mutants were still able to inhibit growth of F. oxysporum f. sp. radicis-lycopersici on plates. Their results clearly showed, for the first time, the crucial role of colonization in biocontrol (Chin-A-Woeng et al. in press).
Colonization mutants of strain WCS365 show a less clear effect (Table 4), because some mutant strains (e.g., PCL1269, PCL1233, and PCL1210) show efficient biocontrol in some, but not all, experiments, whereas strain PCL1209 shows bio-control in all three experiments. We conclude that colonization plays a less important role in biocontrol when strain WCS365 is used than when strain PCL1391 is used. The most likely explanation is the difference in biocontrol mechanisms used by the two strains. In the case of PCL1391, production and secretion of the antifungal metabolite PCN on a substantial part of the root during the whole plant growth period is likely to be necessary to protect the root against pathogens from the soil. In the case of WCS365, which is supposed to act through ISR, it is conceivable that colonization of seedlings for a brief period is sufficient to induce ISR in the whole plant.
Colonization is often the limiting step in biocontrol; there-fore, the importance of the sss gene in colonization (Dekkers et al. 1998b) raised the question of whether this operon can be used to improve colonization in other Pseudomonas spp. Re-sults (Table 5) show that the presence of multiple copies of an sss-containing fragment in the wild-type strain WCS365 has no positive effect on root tip colonization. However, the pres-ence of the same fragment in two P. fluorescens strains, F113 and WCS307, improved root tip colonization enormously. These experiments show that improvement of colonization through genetic engineering is a realistic goal. This suggests that, in cases in which colonization is limiting for biocontrol, the use of col genes can improve disease control.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. All Pseudomonas strains (Table 1) were grown overnight at 28°C on solidified King’s medium B (King et al. 1954) or in
Table 4. Biocontrol activity of Pseudomonas fluorescens WCS365 and its colonization mutantsz
Diseased plants (%)
Inoculated strain Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 Exp. 6 Exp. 7
None 42 a 37 a 63 a 46 a 45 a 43 a 72 a WCS365 26 c 21 b 44 b 36 b 19 b 25 b 39 bc PCL1269 … … … 36 a 42 bc PCL1209 … … … … 16 b 26 b 51 b PCL1233 35 abc 23 b 58 a 47 a 16 b 30 a 47 bc PCL1210 33 ab … … 45 ab 20 b 27 b 37 c
zData are the result of eight replicates of 12 plants each. It should be noted that the methods used for seed inoculation in experiments 1 through 4 and 5 through 7 were different. Data were analyzed for significance using analysis of variance followed by Fischer’s least significant difference test (α = 0.05), using SPSS software (SPSS Inc., Chicago). Values with different letter indications indicate a statistically significant difference.
liquid King B under vigorous aeration. For the transfer of plasmids from Escherichia coli to Pseudomonas spp., tripa-rental mating was performed using pRK2013 as the helper plasmid (Ditta et al. 1980). Selection for plasmid pWTT2081 and its derivatives (van der Bij et al. 1996) was performed on medium supplemented with nalidixic acid and tetracycline to final concentrations of 15 and 80 µg/ml, respectively. E. coli cells used for propagation of plasmids were grown overnight in liquid or solidified Luria-Bertani medium (LB) (Sambrook et al. 1989) supplemented with tetracycline (40 µg/ml). If ap-propriate, X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) was added to the medium to a final concentration of 40 µg/ml as an indicator for β-galactosidase activity.
Colonization experiments on tomato plants.
The ability of various Pseudomonas strains to colonize to-mato root tips was studied using the gnotobiotic system de-scribed by Simons et al. (1996). Briefly, sterile germinated tomato seeds were inoculated with a 1:1 mixture of cells of two strains, one of which was marked with Tn5lacZ. After growth for 7 days, bacteria were isolated from the root tip (1 to 2 cm). When plasmid-containing strains had to be tested, pWTT2081, a rhizosphere-stable plasmid (van der Bij et al. 1996), was used as the vector. This plasmid acts as a genetic or physiological burden (van der Bij et al. 1996); therefore, it also was present in the control cells. The ratio of cells har-boring plasmid pMP5215 (yellow/white colonies) and cells marked with Tn5lacZ and additionally harboring plasmid pWTT2081 (blue colonies) was determined by plating on King B supplemented with X-gal.
Biocontrol experiments.
For experiments 1 through 4 (Table 4), suspensions of ap-proximately 1012 CFU/ml were made by scraping
Pseudo-monas bacteria from King B plates and suspending them into 6 ml of sterile 10 mM MgSO4. These suspensions were used
to coat seeds of tomato (Lycopersicon esculentum Mill. cv. Carmello) in a small-scale industrial fluidized bed coater. For all other seed coatings performed for biocontrol experiments,
overnight King B cultures were washed and adjusted to an optical density at 620 nm (OD620) of 0.7. For coating with a
mixture of both WCS365 and PCL1391, cultures of OD620 of
0.7 were mixed in a ratio of 1:1. All suspensions were mixed with an equal volume of 2% (wt/vol) methylcellulose. Tomato seeds were inoculated by dipping in the resulting suspensions for 10 min and air-dried. Biocontrol by various Pseudomonas spp. against F. oxysporum f. sp. radicis-lycopersici on tomato was performed as described by Chin-A-Woeng et al. (1998). Coated tomato seeds were sown in F. oxysporum-infested soil in multicell trays. For each coating, at least eight replications containing 12 plants each were used. After incubation for 16 days in a climate-controlled growth chamber at 20°C and 85% relative humidity, the number of diseased root systems was assessed. Data were analyzed for significance using analysis of variance followed by Fischer’s least significant difference test (α= 0.05), using SPSS software (SPSS Inc., Chicago). Microscopy.
Germinated tomato seedlings were inoculated with a 1:1 mixture of P. fluorescens WCS365, harboring the DsRed- or ecfp-containing plasmid, and P. chlororaphis PCL1391, har-boring egfp- or eyfp-containing plasmid, and planted in the gnotobiotic system (Simons et al. 1996). After 1 week, the tomato roots were isolated and rinsed in phosphate-buffered saline solution (PBS; 0.9% NaCl buffered with 10 mM so-dium phosphate, pH 7.2). Tomato roots were mounted on a microscope slide for observation. Roots were examined with an inverted fluorescence microscope (Leica DMIRBE; Leica, Bensheim, Germany) equipped with filter blocks with spectral properties matching those of CFP (440/21-nm excitation, 480/36-nm emission, XF114; Chroma, Brattleboro, VT, U.S.A.) or EGFP and EYFP (470/20-nm excitation, 515-nm long pass emission, I3; Leica) or DsRed (538/22-nm excita-tion, 590-nm long pass emission, N2.1; Leica). A Leica SP confocal scanhead was attached to this microscope. Dual color images were obtained by sequentially scanning with settings optimal for CFP (excitation with the 457-nm argon laser line, detection of emitted light between 470 and 490 nm), EGFP (excitation with a 488-nm argon laser line, detection of emit-ted light between 500 and 520 nm), EYFP (excitation with the 488-nm argon laser line, detection of emitted light between 530 and 550 nm), or DsRed (excitation with the 568-nm krypton laser line, detection of the emitted light between 580 and 620 nm.) Cross talk between the channels in this set-up was monitored and, in all cases, was negligible. Pictures ob-tained using different channels were merged using Photoshop 5.0 (Adobe, San Jose, CA, U.S.A.) to facilitate projection. ACKNOWLEDGMENTS
We thank B. A. M. Kroon and R. J. Scheffer of Novartis Seeds B.V. (Enkhuizen, The Netherlands) for performing experiments 1 through 4 of Table 4; and Davy de Witt for his help with biocontrol experiments. C. C. P. was supported by the NWO-LNV Priority Program Crop Pro-tection, project number 805.45.008. The investigations were partly sup-ported by the EU program BIO4-CT98-0254.
LITERATURE CITED
Bloemberg, G. V., Wijfjes, A. H. M., Lamers, G. E. M., Stuurman, N., and Lugtenberg, B. J. J. Simultaneous imaging of Pseudomonas fluo-rescens WCS365 populations expressing three different autofluores-Table 5. Effect of the introduction of the sss-containing plasmid
pMP5215 on the competitive tomato root tip-colonizing ability of vari-ous Pseudomonas fluorescens wild-type strainsy
Colonizing abilityz
Strain Wild-type Wild-type harboring plasmid pMP5215 WCS365 4.4 ± 0.4 a 3.8 ± 0.5 a
F113 3.3 ± 0.8 a 4.5 ± 0.3 b
3.7 ± 0.3 a 4.6 ± 0.4 b WCS307 3.0 ± 0.9 a 4.6 ± 0.2 b 3.3 ± 0.8 a 4.4 ± 0.3 b
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zMean log
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